PERK途径内质网应激与右美托咪定减轻小鼠脑缺血再灌注损伤的关系  

Relationship between PERK pathway-mediated endoplasmic reticulum stress and reduction of cerebral ischemia-reperfusion injury by dexmedetomidine in mice

作　　者：吴科帆 张爱宁 季烨龙 张艺[1] 江梦[1] 夏中元[1] Wu Kefan;Zhang Aining;Ji Yelong;Zhang Yi;Jiang Meng;Xia Zhongyuan(Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院麻醉科,430060

出　　处：《中华麻醉学杂志》2021年第5期546-550,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81801390,81671891)。

摘　　要：目的:从在体和细胞水平评价蛋白激酶R样内质网激酶(PERK)途径内质网应激与右美托咪定减轻小鼠脑缺血再灌注损伤的关系。方法:在体实验健康清洁级雄性小鼠20只,体重20~30 g,6~8周龄,采用随机数字表法将小鼠分为4组(n=5):假手术组(S组)、假手术+右美托咪定组(SD组)、脑缺血再灌注组(IR组)和脑缺血再灌注+右美托咪定组(IRD组)。采用改良二血管阻断联合低血压法制备脑缺血再灌注损伤模型。IRD组于缺血10 min时、SD组于相应时点腹腔注射右美托咪定25μg/kg。再灌注1 h时采用改良神经系统损伤程度法进行行为学评分,随后处死大鼠,取脑组织,采用Western blot法测定内质网应激蛋白:免疫球蛋白重链结合蛋白(BIP)、真核起始因子2α(EIF-2α)、p-EIF-2α、PERK、p-PERK的表达。细胞实验选取标准小鼠海马神经元细胞系,采用随机数字表法分为4组(n=12):对照组(C组)、糖氧剥夺/复氧复糖组(OGD/R组)、糖氧剥夺/复氧复糖+右美托咪定组(OGD/R+D组)、糖氧剥夺/复氧复糖+PERK途径抑制剂ISRIB组(OGD/R+ISRIB组)。糖氧剥夺/复氧复糖模型的制备:使用低糖培养基在94%N 2-5%CO_(2)-1%O_(2)混合气体孵育6 h,然后复氧复糖。于糖氧剥夺前30 min,OGD/R+D组加入终浓度5 mmol/L的右美托咪定,OGD/R+ISRIB组中加入终浓度10 mmol/L的ISRIB。复氧复糖12 h时,采用CCK-8法检测细胞存活率,复氧复糖24 h时,采用Western blot法测定内质网应激蛋白的表达。结果:在体实验与S组比较,IR组行为学评分升高,脑组织BIP、p-EIF-2α、p-PERK表达上调(P<0.05)。与IR组比较,IRD组行为学评分降低,脑组织BIP、p-EIF-2α、p-PERK表达下调(P<0.05)。细胞实验与C组比较,OGD/R组BIP、p-EIF-2α、PERK与p-PERK表达上调,细胞存活率下降(P<0.05);与OGD/R组比较,OGD/R+D组、OGD/R+ISRIB组BIP、p-EIF-2α与p-PERK表达下调,细胞存活率升高(P<0.05);与OGD/R+ISRIB组比较,OGD/R+D组PERK表达下调(P<0.05),其余指�Objective To evaluate the relationship between protein kinase RNA-like endoplasmic reticulum kinase(PERK)pathway-mediated endoplasmic reticulum stress and the reduction of cerebral ischemia-reperfusion(I/R)injury by dexmedetomidine in mice by the in vivo experiment and the cell experiment.Methods In the in vivo experiment,20 healthy clean-grade male mice,aged 6-8 weeks,weighing 20-30 g,were divided into 4 groups(n=5 each)using a random number table method:sham operation group(group S),sham operation+dexmedetomidine group(group SD),cerebral I/R group(group IR)and cerebral I/R+dexmedetomidine group(group IRD).Cerebral I/R was established by two-vessel occlusion plus hypotension.Dexmedetomidine 25μg/kg was intraperitoneally injected at 10 min of ischemia in group IRD and at the corresponding time point in group SD.Neurological function was assessed using modified neurological severity score at 1 h of reperfusion.The animals were then sacrificed and brain tissues were taken for determination of the expression of endoplasmic reticulum stress-related proteins such as immunoglobulin heavy chain-binding protein(BIP),eukaryotic translation initiation factor 2α(EIF-2α),phosphorylated EIF-2α(p-EIF-2α),PERK and phosphorylated PERK(p-PERK)(by Wester blot).In the cell experiment,a mouse hippocampal neuronal cell line was selected and divided into 4 groups(n=12 each)using a random number table method:control group(group C),oxygen-glucose deprivation/restoration(OGD/R)group(group OGD/R),OGD/R+dexmedetomidine group(group OGD/R+D)and OGD/R+ISRIB(PERK pathway inhibitor)group(group OGD/R+ISRIB).Cells were exposed to 94%N2-5%CO_(2)-1%O_(2) and incubated in a low-glucose DMEM medium for 6 h followed by restoration to establish OGD/R model.At 30 min before OGD,dexmedetomidine(final concentration 5 mmol/L)was added in group OGD/R+D,and ISRIB(final concentration 10 mmol/L)was added in group OGD/R+ISRIB.After 12-h restoration was completed,the cell survival rate was detected by CCK-8 assay.At 24 of restoration,the expression of endop

关 键 词：右美托咪啶 脑 再灌注损伤 内质网应激 eIF-2激酶 

分 类 号：R965[医药卫生—药理学]